Alternative Control Measure for Reducing Citrinin and Alternariol in Broad Beans.A

INTRODUCTION:

Faba bean (Vicia fabae L.) is one of the most important food legumes due to its high nutritive value in terms of energy and protein contents (24-30 %),58% carbohydrate and it is an excellent nitrogen fixer (El wakile et al., 2009). Egypt is the second largest producer of faba bean in the world, next to china (Torres et al., 2006a).The annual faba bean production in Egypt is ranging from 0.56 to 0.58 million metric ton during   2003-2005. However, this production is not enough to meet domestic demand (FLRP, 2004, ICARDA, 2005, Maalouf et al.,2009 and yehia et al .,2011). The first report concerning a new disease of faba bean crops which has been invaded by Alternaria  tennuisina  in Japan  (Mohamed et al.,2002 and Barkai-Golan and Nachman,2008).

Alternaria alternata (Fr.) Keissler is a ubiquitous fungi and cause numerous plant diseases and many damages to crops in the field (Mašková et al.,2012). This facultative pathogen induces (spots and lesions) mainly on leaves and fruit and less severely on stolons and finaly leads to complete death of the plant. (El Morsi et al., 2006 and Vûckovíc et al.,2012). This disease was found in several countries and several plant crops (Bodroža-Solarov et al.,2012 and Vûckovíc et al.,2013). In addition to economic losses in production and processing (Kosiak et al., 2004),the disease lead to  significant reduction in grain quality (Bodroža-Solarov et al., 2012)  , this fungus produce AAL toxins (which means Alternaria alternata toxins)with different toxicological properties. The presence of these compounds in the food chain is an increasing concern for human and animal health due to their possible harmful effects. (Fern´andez-Cruz et al., 2010 and Burkin and Kononenko 2011).

There is a wide range of alternaria toxins but only few of them are associated with health risk acute toxicity (EFSA, 2011). AOH (alternariol) and AME(alternariol monomethyl ether ) are of the major concern since they were found to have mutagenic, genotoxic and carcinogenic effects on mammalian cells (Battilani et al., 2009 and Bhaat et al.,2010).

Alternariol was detected in many agricultural commodities and other foodstuffs (Asam etal.,2010 and 2011, Malachova etal.,2011,Suchowilska et al.,2010, Vučković et al.,2012, Eckhout et al.,2013).

Both of Alternaria alternata and penicillium citrinum are notusually broad bean seeds invaders pathogens (Dixit and Singh).According to the available literature, this may be the first registration of this phenomena in Egypt suggesting the possibility to be a new patho-types. 

Citrinin is acutely nephrotoxic at relatively high dose in mice and rats, rabbits ,pigs and poultry causing swelling and eventual necrosis of the kidneys and affecting the liver function at a lesser extents (Abou-Zeid ,2012 and Friedman and Rasooly , 2013). There is no specific fungicides for controlling this crop disease case .However,many attempts in vitro are reported for inhibiting  Alternaria alternata and Penicillium citrinum using metalaxyl and ridomil MZ (Javaid et al.,2006).

Metalaxyl: is used to control certain  diseases caused by air-and soil-borne fungi. Foliar sprays with mixture of metalaxyl and protectant fungicides are recommended to control certain air-borne diseases.

Ridomil MZ 72 WP contains metalaxyl 8% and mancozeb 64%. Recently Shu-Yuan Cheng et al., 2014 demonstrated that  mancozeb (MZ) induced cytotoxicity in rat pheochromocytoma (PC12) cells partially via nuclear factor kappa B (NF-κB) activation. This study demonstrated that MZ induced DNA damage as seen in comet assay.

Zeidan, (2006) reported that benzoic acid was applied on grapevine in Germanic green houses as MENNO-Florades (9% benzoic acid) to antagonize Plasmopara viticola.This application realized highly antagonistic frequency (91.40%), ( two days before fungal infection).

 Therefore, the aim of this study is to reduce the applied dose of fungicides by using preservatives and investigate their side effects on mycotoxins detoxification under field conditions.

Materials and Methods

Fungicides:

Metalaxyl: (IUPAC) name:  methyl N-(methoxyacetyl)-N-(2,6-xylyl)-D-alaninate.            

Ridomil MZ: methyl N-(methoxyacetyl)-N-(2,6-xylyl)-D-alaninate+ Mancozeb: manganese ethylene bis (dithiocarbamate) (polymeric) complex with zinc salt.

Organic acid used as a food preservatives:

 Benzoic: C₇H₆O₂ (or C₆H₅COOH).

 Sorbic acid, or 2,4-. hexadienoic acid: C₆H₈O₂

Structures of mentioned Mycotoxins:

 Alternariol is 3,7,9-Ttrihydroxy-1 methyl-6H-dibenzo[b,d] pyran-6-one.Its molecular formula C₁₄H₁₀O₅ .

Citrinin [C₁₃ H₁₄ O₅₁] IUPAC: (3R, 4s)-4,6- dihydro-8nhydroxy-3,4,5-trimethyl-6-oxo-3H2- benzopyran-7-carboxylic acid.

 The seed borne fungi associated with infected broad bean seeds collected from Noubaria region are previously isolated, purified and identified from surface sterilized seeds using seed health testing method and Frequency of the obtained fungi (Alternaria alternata ,Penicillium citrinum and Aspergillus flavus) was determined. Moreover, mycotoxin production ability was estimated and the occurred mycotoxins (alternariol and citrinin ) were detected and registered as mentioned in previous study (Youssef et al., 2014).

Preparation of chemicals and organic acids serving in Agricultural Experiment:

Benzoic and sorbic acids used as chemical or synthetic preservatives (Class II) are brought from Sigma, Aldrich, Bureau of Cairo Egypt. The food standards regulations required that not more than one Class II preservative should be used in one particular food item (Anand and Sati, 2013). Metalaxyl and ridomil MZ are brought from Syngenta Agro Egypt manufacturing.

 Agricultural experiment

  Preliminary tests for agricultural experiment:

It was necessary to test the performance of the used tested treatments on seed germination process, and fungal growth. The percent germination process was considered as the determining factor of how tested treatments will be applied during agricultural experiment either seed treatment before seeds Sown process or spraying seedling with the tested treatment every two weeks during the experiment (first test). Furthermore, it was necessary to determine the effect of these treatments on the growth of the occurred fungi associated with infected seeds (second test).

First test: Effects of the tested treatments on seed germination process .

Untreated non infected seeds (Giza blanka) were brought from Research Seeds and Oily seeds Institute, Agricultural Researches Centre, Giza, Egypt. Germination viability test was carried out according to ISTA (1996), Singh and Garampalli, (2012) and Soughir et al., 2012 with certain modification. 100 g of seeds were taken from each healthy and/or infected seeds and divided into 5 replicates - 20g each. Two fungicides metalaxyl and ridomil MZ treatments are used at concentrations 0.5 x, x and 1.5 x (where x =500mg /250ml water). Benzoic and sorbic acids and their esters are used at concentrations 5, 10, 15 and 25 ppm. The effect of ethyl alcohol on germination process at concentration 0.25% was tested also.  Seeds (20g) in each treatment were placed in sterilized cup 125 ml in volume, the tested concentration was added to sterilized distilled water 100 ml to each cup for each treatment. The negative control for both healthy and infected healthy seed replicates comprise distilled sterilized eater treatment. Each cup solution was decanted 5 hours later, and then weighed.  Seeds from each treatment were placed in 120-mm-diameter Petri dishes between two layers of Watman filter paper and then moistened with 10 ml of distilled water for negative control, whereas, the other treatments were  moistened with the same solution of the tested treatment . Seeds were kept at room temperature (25°C) under normal light. The number of germinated seeds was counted daily for 7 days after which no further seed germination was occurred. The appearance of 2 mm or more of radical was considered as germination. Parameters measured in this experiment were registered:

Total germination (TG) measured in the seventh day using the formula GT (%) = (total number of germinated seeds (B)/ total seed (A) x 100.( Scott et al.,1984 and Moradi et al., 2008). The obtained data were registered in Table (1).

Second test 2 :( Confirmation):  Testing  the treatments effect on growth of fungi occurred in seeds:

 The aim of this test was to determine the suitable concentrations for each treatment which will be applied during agriculture experiment. 15 seeds per replicate were taken then soaked in different concentrations of the tested fungicides and preservatives for 10 minutes. Healthy seeds for control were initially surface sterilized using sodium hypochlorite solution 5.0 % for 3 min. and then they were rinsed 3 times with distilled water for 2 min. (Soughir et al., 2012). For non infected and naturally infected control, the seeds were soaked in distilled water. Seeds of all the treatments were placed on filter papers in 9 cm diameter petri plates, moistened with 3 ml of distilled water and/or tested treatment concentration each. Plates were incubated in a growth room at 25±2°C for 8days under alternative light and dark 12 hours each. Each treatment was replicated thrice (Christensen, 1957; Javaid et al., 2006 and Sitara and Hasan, 2011 with certain modification).

After 8 days of incubation, fungal species grown on the surface of treated seeds were purified and identified and their percentage frequency (PF) was determined according to Samson et al. (1996) and Javaid et al. (2006). The number of colonies growing on each plate was counted for each identified fungus and its frequency percentage was calculated according to the following formula (Pitt and Hocking, 1997):

The resulted data were registered as shown in Table ( 2). The ability of the main occurred fungi was previously tested and detected as mentioned in precedent study (Youssef et al., 2014).The resulted data were registered as shown in Table (2). The selected treatment concentrations for agricultural experiment were summarized in Table (3).

Agricultural experiment:

The clay soil was used in this study ,the electrical conductivity(EC), in decisiveness per meter was measured by Baxter, Digital conductivity meter in saturated soil paste extract as described in hand book 60 (U.S Salinity Laboratory Staff, 1954). Acidity (pH value) was also determined in the tested soil paste using pH meter.

Plastic pots containing 1.5 kg sterilized autoclaved clayey soil for 30 min. and kept covered until sowing seeds. Both of healthy and infected seeds were surface sterilized using sodium hypochlorite 5% for 2 min.  Seeds were then rinsed three time in 100 ml sterilized water each to completely get rid of sodium hypochlorite. Weighed seeds for 1hour (10 seeds per pots) were carefully sown. Each treatment was replicated thrice. Pots were placed in complete sunny and fresh air weather to accomplish the field conditions. Seedlings of two weeks age are sprayed with tested treatments. The spraying process was applied and repeated fortnightly.At the end of the experiment, the number of dead plants was counted, measured as illustrated in Table (4), plants, 60 days age, were collected, washed then air dried.  Plant fresh weight, root and shoot lengths and shoot black lesions lengths (for both dead and alive plants) were measured. Data were registered as illustrated in Table (5).

Estimation of the inhibitory effect of treatments on plant contents of alternariol (AOH) and citrinin (CTN):

The main target of this experiment was to achieve the best treatment which reduce or prevent the production of the two tested mycotoxins during agriculture experiment (in vivo) under field conditions.  Moreover,this experiment was aimed too to study the following:-The transition of  alternariol (AOH) and citrinin (CTN ) during the agricultural process from sown seeds to plant; and the effectiveness of the applied spraying method in reducing the two tested mycotoxins.

Plants in each treatment were gently cut into small pieces, and then puted in sterilized cups 10 g each. Treatments were triplicated. Plant samples were prepared to be detected firstly by using TLC ( Thin layer chromatography) plate (Jouany, 2007 and Kütt et al., 2010).Two development systems were used for each toxin: Anis: 0.5% p-anisaldehyde in methanol/acetic acid/conc; and sulphuric acid (17:2:1v/v/v) for alternariol and benzene: glacial acetic acid: acetone (5:4:1) v/v/v (Frisvad et al. 2007 and Eckhout et al., 2013). Under UV light at 254 nm AOH was detected whereas, CTN was detected at 366 nm. After that the detection of alterniol (alternariol) and citrinin was confirmed by using quantitatively HPLC-UV technique according to Azcarat et al., (2008), Asam et al. ,(2011) and Brzonkalik et al., (2011) at Central Lab, Faculty of Pharmacy, Alexandria University. The obtained citrinin and alternariol standards were brought from Sigma, Aldrich ,Cairo, Egypt. Results were registered in Table (4).

Statistical analysis:

The experiment was laid out in completely randomized design with three replications. The data were subjected to statistical analysis using Costat computer package (CoHort Software, Berkeley, CA, USA). Least significant difference (LSD) using Duncan’s Multiple Range test was applied to compare the treatment mean values according to McDonald, (2009).

Results and discussion

2. a. Determination of the Electric conductivity and pH in soil used in the experiment.

The applied soil was sterilized before planting and soil electrical conductivity (EC) and pH degree were determined.  EC of the soil paste was 3.4 dS/m, whereas soil pH was 7.5.

2. b. Tested treatments effects on seed germination process .

The germination viability of seeds under treatments concentration was tested. Resulted data as illustrated in Table (1) indicated the following: The number of seeds in the same weight varied according to fungal infection. Furthermore, the degree of infection varied between seeds in the same yield. Germination process was affected by fungal infection, the average of germinated seeds attained the highest value at metalaxyl at recommended dose (x) and ridomil MZ at (1/2x).

Retardation in germination was observed during treatment with ethyl alcohol 25%, but it was completely inhibited and delayed in treatments, treated with the tested preservatives and their esters. Fungal infection reduced seed germination rate. These findings were totally in harmony with those reported by Embaby et al. (2013) and Perelló and larrán (2013). These results limited the mode of treatments application as seedling spraying method. Both of benzoic and sorbic acids and their esters failed to be applied as seed treatment. Our results are completely in agreement with those of Maouni et al. (2002), Sitara and Akhter (2007), Saleem et al. (2012) and Sarmamy and Khidir, (2013), who reported that ridomil MZ , ridomil MZ gold and metalaxyl increased seed germination percentages. Moreover, results obtained by  benzoic acid and other alternatives were totally in agreement with Sunaina and Singh ,(2014) and indirectly coincided  with those of Yadav and Singh (2013),  who reported that benzoic acid (BA) decreases the germination ratio of Triticum aestivum L. sown in pots containing concentrations (0.5,1and 1.5 mM) of BA in dose dependent manner. Maximum 37.5% reduction in germination was recorded at highest concentration of BA. In our study benzoic, sorbic acids and their esters completely failed in germination process.  Differences may be attributed to differences in seeds cultivars, and s the applied method. In our experiment, faba bean seeds were completely emerged in aqueous solution in treatment left for 5 h. before decanting water then placed between two filter papers imbibed with the tested treatment concentration. Thus, the treatment was entirely  available for seeds, whereas in Yadav and Singh experiment (2013), seeds were sown in soil containing high concentration of benzoic acid each but undoubtly,  the bioavailability of treatment  in aqueous media is different than soil because soils can absorb this acid, consequently reduce the treatment bioavailability to seeds. Our findings were also in compatible with those reported by Deepavali and Nilima ,(2012), who reported that aflatoxin decreases germination rate.

Second test: Inhibitory Effect of the treatments on fungal growth of seed flora:

The effects of the tested treatments on growth of fungal seed flora were confirmed. Treatments have inhibitory effects against Alternaria alternata, Penicillium  citrinum ,Aspergillus flavus, Penicillium sp and Aspergillus niger. Results were registered as elucidated in Table (2).

Metalaxyl had moderate inhibition against A. alternata, but  increased the Penicillium citrinum growth. Our findings were highly in accordance with those of Cohen, (1981), who found that metalaxyl enhanced the penicillium growth in citrus fruits. Our results were also harmoniously compatible with those of Matheron and Porchas (2000) and Saleem et al. (2012), who reported that metalaxyl 15% + copper oxychloride and ridomil MZ (metalalaxyl+mancozeb) exhibited a satisfied inhibitive effect against A.alternata  and P.expansum. According to   results obtained in the present investigation and those reported by others, chemicals added to metalaxyl such as copper oxychloride or mancozeb  ameliorate augmented the inhibitory effect of these mixture against Penicillium than  metalaxyl  alone.

Agricultural experiment:

2.1. Determination of death rate:

The aim of this test is to determine the effect of fungal infection on plant death and investigate the effects of the tested treatments on reducing death rate. Data in Table (4) illustrated that ridomil MZ at conc. 1.5 x realized the best results in maintained plant life under fungal infection conditions, followed by benzoic acid at 10 ppm. Sorbic acid at conc.10 ppm and its ester form revealed approximately the same action. These findings were partially in harmony with Hawthorne et al. (2014), who reported that mancozeb (750-800g/kg) significantly suppressed  Alternaria spp growth, compared with the other tested  fungicides. Similarly, the findings of the same authers may also explain our second finding concerning the worth treatment in maintained plant life enhanced by metalaxyl and ridomil MZ at their recommended dose. This notice may also indicated that these recommended doses were applied only for healthy seed and  plant protections but not for actually occurred infection treatment. Death rate in untreated infected plant was extremely high. Our findings are harmoniously agreed with those of Perelló and larrán (2013), who reported that Alternaria not only reduces germination and vigor of wheat seed but it also causes seedling blight disease in Argentina (Rajput et al., 2005 and Perelló, 2010 a and b) its transmission from seed to seedling of wheat seeds has been done in Argentina. Moreover, every year this seed-borne fungi cause heavy yield loss of the crop.

Table (1): Comparing between the influence of certain fungicides, organic     acids and esters on germination process of broad bean seeds

Where:

-T.S.N /W/5replicate= Total seed number  in 20g seeds per 1 replicate.

*= ethyl alcohol concentration-

- used = 1: 4 sterilized de-ionized water.

**= The germination process started  later than the other treatments.

  X=recommended dose for application

 (R.D) = 0.5g/250ml water).                                                     

 G start = day in which seed start to  germinate.

  H. seeds = non infected seeds.

Table (2):  Effect of the tested treatments on fungal inhibition

     Where: 8*= 6 colonies of Penicillium sp and 2 colonies of Aspergillus niger & PF = Frequency percentage of each fungal sp.& 2*+2**= 2*Penicillium sp colonies+   2**A.flavus colonies &       ** = A.flavus colonies

    Our selected treatments are summarized as shown in Table (3)

Table(3): Treatment concentrations applied in the Agricultural experiment.

Table (4): effect of the tested treatments on reducing plant death:

2.2. Effects of the tested treatments on certain plant growth parameters:

Seedling shoot and root lengths, plant fresh weight, black color lesion (BCL) on faba bean seedling shoots were determined.  BCL ratio was calculated for each treatment to assess the effects of the tested treatments on curing infected seedlings. Data in Table (4) illustrated that fungal infection significantly reduced all plant growth parameters and realized the highest BCL values, which indicated the relation between BCL produced by these pathogens and their virulence in field. These findings were in harmony with Yamaji et al. (2001); Khan et al. (2008) and Hawthorne et al. (2014).

Our results concerning the effect on shoot length revealed that Ridomil MZ realized the two highest shoot lengths, followed by ethyl benzoate at conc.5 ppm then ethyl sorbate. Metalaxyl and benzoic acid decreased shoot length These findings were in agreement with Wandrey et al. (2004), who mentioned that metalaxyl affected plant growth and plant height only when was applied as spraying. Moreover, these results coincided with those reported by Maffei et al. (1999); Yadav and Singh (2013) and Sunaina and Singh (2014),  who reported that benzoic acid (BA) significantly decreased plant growth and seed germination proportionally with the increase in concentration.

Concerning root length, ethyl benzoate, ethyl sorbate at 5 ppm and sorbic acid at 10 ppm realized the best same action as  ridomil MZ at 1.5X on root length. In contrast to that, metalaxyl, benzoic acid and sorbic  acid at 10 ppm  achieved the worth results. These results are partially in agreement withMagarey  et al. (1995 and 1997) and Matheron and Porchas (2000),  who mentioned that mancozeb ameliorate root and shoot growth  than metalaxyl and were in harmony with those of Fujita and Syono (1996), who reported that the extent of inhibition of root growth by benzoic acid  at 10 / µMin pir2-l and pir2-l auxl-7 seedlings was 44% and 19%, respectively as compared with  control in absence of  benzoic acid (BA) without any inhibition of polar auxin transport (Hertel  et al., 1969 and 1983). On the other hand, Kim and Roh (2014) reported that BA appeared to promote the growth of tobacco plants. The best growth was obtained on day 50, but the activity of the antioxidative enzyme (glutathione reductase) was inhibited, which suggested that faba bean plant is less resistant to BA than tobacco plants.

Seedling fresh weight determinations :both of ethyl benzoate (5ppm) and sorbic acid (10ppm) realized similar effects as ridomil MZ (1.5x) on seedling fresh weight. Our findings were in agreement with those of Magarey and Bull (1994) and Saleem, (2010), who reported that mancozeb augmented shoot and root dry weight of sugarcane. Furthermore, Magarey and Croft (1995) reported that metalaxyl reduced shoot and root dry weight. Moreover, Magarey and Bull (2003) found that mancozeb caused yield decline in affected soil.

Black color lesions (BCL) attained the highest values in case of infected untreated seedlings and was absent in uninfected seedlings. These results indicated that the existence of this phenomenon was completely correlated to phytopathogenicity and plant diseases. According to many literatures, the seedling infection with Alternaria alternata or Penicillium citrinum do not produce these large black color lesions but the last one appeared when the double infection with both fungus occurred.  Thus, it is suggested the presence of a synergistic effect between the two last mentioned fungi which appeared strongly on BCL production is a sort of virulent phytopathogenisty. Our findings are coincided with those of Perellَ et al. (2005, 2008 and 2012 and Benssassi et al. (2009).

Treatment’s effectiveness rank was as the following: ethyl benzoate at 5ppm > benzoic acid at 10pm > ridomil MZ at 1.5 x > sorbic acid at 10ppm then ethyl sorbate at 5ppm. Both of benzoic and sorbic acids at 15 ppm had approximately the same efficiency of metalaxyl x.

2.3.Effect of the  tested fungicides and their  alternative preservatives on alternariol (AOH) and citrinin (CTN) inhibition in obtained plants after agriculture process.

This experiment was carried out on plants collected and prepared for mycotoxins estimation as mentioned above and data were registered and illustrated as shown in Table (5).

Concerning transition of AOH and CTN during the agriculture process from sown seeds to plant, according to Youssef et al. (2014), the average of seeds mycotoxins contents   in non-treated seeds were 231.00 µg/gm for AOH and 337.00 µg/g for CTN (Table 6). It was remarked that during agriculture process both of the tested mycotoxins were transmitted from infected seeds to infected plants containing 219.739 and 250.950 µg/g in AOH and  CTN, respectively. These findings are coincided with those of Muller and Amend (1997) and Perelló and larrán (2013), who suggested that the decline in mycotoxins degradation in plant was resulted from the exhaustion of nutrients lowering the mycotoxin production rate. Furthermore, our results are compatible with those of Miller et al., 1983 and Bhatnagar et al. (1991), who hypothesized that the biological degradation of mycotoxins in plant is an enzymatic degradation which considered the plant as source of these enzymes. Lugauskas, (2005) mentioned that AOH and CTN need a sort of fermentation to be degraded.

The efficiency of the tested treatment in inhibiting or reducing AOH and CTN during agriculture process: Ethyl benzoate at conc.10 ppm was the best treatment, where complete inhibition of AOH and CTN production, followed by the concentration 5 ppm then ethyl sorbate  at 10 ppm  and benzoic acid  at 10 and15 ppm, followed by sorbic acid at 15 ppm. Metalaxyl at its recommended dose has a strong inhibitory effects on CTN only but  ridomil MZ   has a quite effects on  both  toxins. The inhibition rate was reversely proportional to concentration in ridomil MZ. These findings were reasonably compatible with those of Abd-El Ghany and Tayel (2009), who reported that fungal growth inhibition was sometimes inversely proportional to mycotoxin inhibition .In contrast to that, sorbic acid at 10 ppm has an effective inhibitory effects on CTN and moderately effect on AOH.

The effectiveness of the applied spraying method in reducing the two tested mycotoxins: According to our obtained results, the applied spraying method during this experiment was very satisfactory effective in reducing the two tested mycotoxins. This explained its success in making the pre-harvest crop’s yield safety for both human and animal consumption and the plant commodities too.  

According to Table (5) and (6) we can notice that in case of BCL, AOH and CTN inhibitions, ethyl benzoate had the inhibitoriest effect.  It can be suggested that both mycotoxins are involved in BCL appearance and phytotoxity. Such explanation was in agreement  with those of Wang (1948);  Nickell and Finlay (1954); White and  Truelove (1972); Damodaran et al. (1975) and Yamaji et al. (2001), who concluded that citrinin at 125 ppm caused collapse of the root structure and tissue browning, and the growth of seedlings was inhibited markedly .

Conclusion:

According to the obtained results, benzoic , sorbic acids and their ester forms especially ethyl benzoate are very effective in reducing the production of AOH and CTN by Alternaria alternate and Penicillium citrinum. The use of ethyl benzoate as alternative to the tested fungicides may realize a big challenge in reducing fungicides used for treatment of infected seedlings as spray. Besides, it reduces the phytotoxic effect of these mycotoxins which lead to diminish the virulence of these pathogens in field.

On the other hand, it was obvious that in order to avoid the failure of benzoic and sorbic acids and ethyl sorbate to realize best growth to the infected seedlings, further studies are needed on treatment of the infected seeds with metalaxyl (x) or  ridomil MZ(x) before sowing seeds and reduction of the number of spraying  time of seedlings with benzoic or sorbic acids or ethyl sorbate by spraying seedlings each 21 days instead of 15 days.

 Table (5): Effect of the tested treatments on certain plant growth parameters: 

Where:

A=Concentration of tested treatments.

B= Average of one seedling shoot length in cm .

C= Average of Fresh weight ofone seedling 

D= Black color lesions(BCL) On shoot in cm .

E= BCL ratio on shoot =black lesions length= Av of  lengths of black lesions on treated seedlings/Av of total seedlings lengths

X=R.D= 500mg/250ml water.

1.5 X= R.D + 0.5 R.D=750mg/250ml & Seedling = complete growing plant but before flowering then fruiting stages.